1. Field of the Invention
The invention relates to a regulatory sequence which mediate a specific expression in liver cells. Moreover, the invention relates to the use of the regulatory sequences.
2. Description of Related Art
α-fetoprotein (AFP) is a plasma fetal protein, the expression of which is primarily restricted to developing tissues of endodermal origin (yolk sac, fetal liver, and gut), although the level of its expression varies greatly depending on the tissue and the developmental stage. AFP is of clinical interest because the serum concentration of AFP is elevated in a majority of hepatoma patients, with high levels of AFP found in patients with advanced disease. The serum AFP levels in patients appear to be regulated by AFP expression in hepatocellular carcinoma but not in surrounding normal liver. Thus, the AFP gene appears to be regulated to hepatoma cell-specific expression.
Previous studies have revealed a complex regulatory mechanisms to promote the temporal and tissue-specific expression of the AFP gene (Wen et al. Nucleic Acids Res. 21: 1911–1918, 1993; DNA Cell Biol. 10: 525–536, 1991; Groupp et al. J. Biol. Chem. 269: 22178–22187, 1994). α-fetoprotein gene expression was regulated not only via a promoter, but also an enhancer. The enhancer, a cis-acting transcriptional regulatory element, is typically characterized by its ability to augment transcription over a long distance and relatively independently of orientation and position with respect to its respective gene to be acted on.
Some studies have been showed that the 5′-flanking region of the human AFP gene contains transcription regulatory elements with characteristics of enhancers (Watanabe et al. J. Biol. Chem. 262: 4812–4818, 1987; CA. Pat. Appl. No. 2,134,994).
In the study of Watanabe et al. (J. Biol. Chem. 262: 4812–4818, 1987), they examined a cis-acting regulatory function associated with the upstream region of the human AFP gene by assaying transient expression of the CAT gene supported by the AFP's 5′-flanking sequence. These result indicated that the region between −5.1 and −2.9 kb of the AFP 5′-flanking sequence is important for the enhancement of CAT expression in HuH-7 hepatoma cells. These result also showed a 7.5 kb fragment and a 0.4 kb fragment from −3.7 to −3.3 kb of the AFP 5′-flanking sequence were both exhibiting cell specificity.
According to the report of Yoshitake Hayashi et al. (J. Biol. Chem. 267: 14580–14585, 1992) and the disclosure of US Pat. Pub. No. 2003/0017139, the −1789 to −1773 bp human albumin 5′-flanking exhibited enhancer activity as well as liver cell specificity. Based on the experimental result of the present invention, however, a pALB2.0 fragment (−1954/+39 bp) comprising the above-mentioned sequence exhibits stimulatory activity in hepatoma and nonhepatic cells, indicating that pALB2.0 fragment has lack of liver cell specificity. Therefore, it is unable to predict that the ability of liver cell specificity can still remain in different length of a known sequence with liver cell specificity. Therefore, there are few application researches about sequences with liver cell specificity.